The successful transfer of embryos was first reported in 1891 (Heap, W., (1891) Proc. R. Soc. 48, 457-458). Since that time, despite numerous advances in assisted reproductive technologies, the assessment of oocyte and embryo quality has changed very little. Morphologic assessment of oocyte and embryo quality has been recognized as a poor indicator of pregnancy potential (Hardy, R. et al, (1993) Am. Fertility Soc. Annu. Mtg., abstract). Significant improvements in ovulation induction, oocyte retrieval, and in vitro culture techniques have resulted in an abundance of embryos per patient or experimental animal. The embryologist must decide which embryos are the highest quality for transfer, yet lacks the means to objectively define oocyte and embryo quality. Choosing the "right" embryo has been deemed the challenge of the nineties (Plachot, M., (1989) J. IVF-ET 6(4), 193-194).
With the advent of invasive and experimental manipulations of the oocyte and embryo, the need for quality assessment has become increasingly important. Considerable efforts have been invested to determine the potential effects of chorionic villus sampling (Jackson, L. et al, (1992) N. Engl. J. Med. 327(9), 594-598). Despite the advent of pre-implantation cytogenetic diagnosis (Coutelle C. et al, (1989) Brit. Med. J. 299(6690), 22-24), however, the effects of embryo biopsy have been less systematically studied. This is in large part due to the absence of an efficient tool for measuring embryo quality.
The ability to select embryos for uterine transfer or to evaluate the effects of such interventions as pre-implantation embryo biopsy, zona drilling, the use of co-culture techniques, and the experimental use of growth factors or cytokines is markedly limited by current techniques, which utilize morphology or calculated percentages of embryos achieving blastocyst development to assess embryo quality.
Many investigators have recognized the need to develop better techniques to assess oocyte and embryo quality. Proposed methods include a development rating system (Cummins, J. et al, (1986) J. IVF-ET 3(5), 284), fluorescein diacetate fluorescence measurement (Mohr, L. et al, (1980) J. Reprod. Fertil. 58, 189), determination of immunosuppressive activity in pre-implantation culture media (Clark, D. et al, (1989) J. IVF-ET 6(1), 51-58), and measurement of immunoactive factors in culture media including progesterone (Hardy, R. et al, (1993) Soc. Gyn. Invest. Annu. Mtg., abstract), interleukin-1-alpha (Hardy, R. et al, (1993) Am. Fertility Soc. Annu. Mtg., abstract) and interleukin-1-beta (Baranao, R. et al, (1992) Am. Fertility Soc. Annu. Mtg., abstract). All of the proposed techniques have significant limitations, and none is in common use.
The metabolism of pre-implantation embryos has been studied extensively (Biggers, J. et al, (1973) Adv. Reprod. Physiol. 6(0), 1-59; Biggers, J. et al, (1967) Proc. Natl. Acad. Sci. U.S.A. 58(2), 560-567). Renewed interest in the field was prompted by the advent of ultramicrofluorometric technology, which allowed the non-invasive measurement of nutrient uptake (Leese, H. et al, (1984) Anal. Biochem. 140(2), 443-448; Gardner, D. et al, (1986) Hum. Reprod. 1, 25). Glucose and pyruvate uptake, lactate production, and purine utilization have been indirectly quantified using traditional enzymatic analysis based on changes in the concentration of fluorescent NADH in culture media (O'Fallon, J. et al, (1986) Biol. Reprod. 34, 58; Gardner, D. et al, (1987) J. Exp. Zoology 242, 103). The measurement of nutrient uptake has also been carried out in human embryos (Leese, H. et al, (1986) Hum. Reprod. 1, 181; Gott, A. et al, (1990) Hum. Reprod. 5(1), 104-8). It is important to distinguish indirect measurement of NADH in culture media from the direct measurements of NADH within oocytes or embryos. Although an apparatus for measuring the state of oxidation-reduction of a living organ (U.S. Pat. No. 4,449,535, issued to Guy Renault) and more specifically, for monitoring the NADH redox state of brain tissue (WO 92/12705, applicant Mayevsky, A. et al) have been described, there have been no direct measurements of NADH within oocytes or embryos.
Although measurement of nutrient uptake has greatly increased our understanding of embryo metabolism, the general applicability of such analysis is limited because the embryos must be cultured in nanoliter-scale microdrops of media. Current practice for clinical and experimental embryo culture prior to embryo transfer requires a substantially larger volume of culture media; this requirement results in excessive dilution of the NAD(P)H concentration and diminution of measurable NAD(P)H fluorescence in the culture media.
In addition to the problem of measuring nutrient uptake by clinical and experimental oocytes and embryos, the fate of nutrients taken up by the oocyte or embryo (i.e., whether the nutrient in fact enters a productive metabolic pathway within the cell), and the predictive value of the uptake studies with respect to mitochondrial function, have not been established. For example, a recent study reported that pyruvate uptake by human embryos was not predictive of those that successfully implanted (Conaghan, J. et al, (1993) J. Assist. Reprod. Genet. 10(1), 21-30).
In summary, current means for assessing oocyte and embryo viability are inadequate and contribute to the low pregnancy rates of assisted reproduction. At present, on average, only 15% of couples who undergo in vitro fertilization (IVF) procedures have a successful pregnancy outcome. Similar problems exist with large animal transgenic procedures. Invasive manipulations of the oocyte and embryo are currently carried out with unknown consequences to the effect of such manipulations on embryo quality.